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目的探讨K-ras突变多肽致敏树突状细胞(DC)对细胞趋化因子CCL19、CCL22和细胞骨架蛋白fascin-1表达的影响。方法联合应用重组人粒-巨噬细胞集落刺激因子和白细胞介素(IL)-4诱导培养外周血DC,收集培养7d后的DC并分为未负载组(加入RPMI1640培养液50μg/ml)和K-ras负载组(加入K-ras突变多肽50μg/ml).流式细胞仪测定负载前、后DC表面标志CD1a、CD80及CD86;扫描电镜和透射电镜观察负载K-ras突变多肽后的DC形态结构;ELISA法检测2组DC培养上清液中IL-12、CCL19和CCL22的表达;Westernblot法测定2组DC骨架蛋白fascin-1的表达。结果①K-ras突变多肽负载DC后,CD1a、CD80及CD86表面分子表达率明显高于负载前(P<0.01).②扫描电镜下见负载后的DC呈花瓣状、树枝样突起;透射电镜下见负载后的DC形态非常不规则,树枝状或毛刺状的突起明显增多。③K-ras负载组负载后不同时相(6、12、24及48h)的IL-12、CCL19及CCL22的表达水平明显高于未负载组(P<0.01).④K-ras负载组fascin-1蛋白的表达水平也高于未负载组(P<0.01).结论K-ras突变多肽能够促进DC成熟,并使细胞趋化因子和骨架蛋白表达水平增加,能够增强DC游走迁移。
Objective To investigate the effect of dendritic cells (KD) stimulated with K-ras mutant on the expression of chemokines CCL19, CCL22 and fascin-1. Methods DCs were induced by recombinant human granulocyte-macrophage colony stimulating factor and interleukin-4 (IL-4). DCs cultured for 7 days were collected and divided into three groups: unloaded group (50μg / ml RPMI1640 medium) K-ras loaded group (50μg / ml K-ras mutated peptide), the surface markers of CD1a, CD80 and CD86 were measured by flow cytometry before and after loading; the morphological changes of DCs bearing K-ras mutant were observed by scanning electron microscopy and transmission electron microscopy The morphological structure was detected by ELISA. The expression of IL-12, CCL19 and CCL22 in DC culture supernatants was detected by ELISA. The expression of fascin-1 was detected by Western blot. Results ① The expression of CD1a, CD80 and CD86 on the surface of CD1a, CD80 and CD86 was significantly higher than that before loading (P <0.01) after DCs were loaded with K-ras mutant. (2) DCs showed petaloid and dendritic processes under SEM; See DC morphology after loading is very irregular, dendritic or burr-like protrusions significantly increased. ③ The expression levels of IL-12, CCL19 and CCL22 at different phases (6, 12, 24 and 48h) in K-ras loaded group were significantly higher than those in non-loaded group (P <0.01) (P <0.01) .Conclusion K-ras mutants can promote DC maturation and increase the expression of chemokines and cytoskeleton proteins, which can enhance the migration of DCs.